Pollen fertility causes problems in various aspects of agriculture and horticulture. For example, in the case of mating for cross breeding, self-pollination has to be avoided by castration (removal of stamens) which requires enormous effort. In the seed and seedling industry, there is a demand for a trait of lack of pollen fertility from the standpoint of commercially protecting excellent breeds obtained by cross breeding. To meet such a demand, a technique for controlling pollen fertility (pollination control) has been strongly required. Conventionally, for particular crops, lines of cytoplasmic male sterility have been used for cross breeding, and some success has been achieved. However, the cytoplasmic sterility trait is often accompanied by undesired side effects, such as a reduction in disease resistance and the like. There are further problems, such as that the trait is unstable, that it is difficult to mass-produce the seeds, and the like. A method for reducing the fertility by treating with a chemical agent(s) has been studied, but safety evaluation and elucidation of the mechanism of this method have not been fully done and thus such a method is not yet in actual use. Therefore, there is a demand for an excellent male sterilization technique using genetic engineering.
Pollen is the male gametophyte of spermatophyte. The development of pollen which proceeds while pollen is surrounded by an anther as a supporting tissue is divided into the following stages: the tetrad stage immediately after the meiosis of microsporogenous cells (pollen mother cells); the release stage during which microspores are released from the tetrad; the uninucleate stage characterized by the enlargement and vacuolation of pollen cells, the mitotic stage giving rise to the differentiation into vegetative and generative cells by mitosis; and the subsequent binucleate stage. After these stages, the anther finally dehisces and matured pollen grains are released. Therefore, it can be said that the microspore is one of target tissues which are most suitable for artificial control in order to inhibit the development of pollen and eliminates pollen fertility.
As described above, great expectations are placed on male sterilization techniques using genetic engineering. Particularly, if a gene which is expressed specifically in the direct precursor of a pollen cell, such as a microspore, can be utilized, it is considered to be highly likely that male sterilization can be achieved without conferring undesired traits to plants. Several examples of promoters specific to various stamen tissues and gene constructs for male sterilization comprising the promoter have been reported (Shivanna and Sawhney Ed., Pollen biotechnology for crop production and improvement (Cambridge University Press), pp. 237–257, 1997). However, there has been continuously a demand for a novel gene useful for control of pollen fertility, which has high tissue and temporal specificities of expression.
Recently, the inventors of the present application specified the cDNA sequences of novel transcription factors derived from Petunia, i.e., seven zinc finger (ZF) transcription factors including PEThy ZPT2-5, PEThy ZPT3-1, and PEThy ZPT4-1 (hereinafter abbreviated as ZPT2-5, ZPT3-1, and ZPT4-1, respectively). And the inventors reported that Northern blot analysis indicates that each transcription factor transiently expresses in an anther-specific manner in a different stage of the development of the anther (Kobayashi et al., Plant J., 13:571, 1998). However, the physiological function and action of these transcription factors in plants, and the precise expression sites and the expression controlling mechanism of the genes encoding the transcription factors have been not clarified.